Alzheimer's disease (AD) is a common chronic progressive neurodegenerative disease in which there is an irreversible loss of cognitive and behavioral functions. The disease can persevere for over 10 years, advancing from mild symptoms to extremely severe manifestations. AD is said to afflict approximately 10% of the population over the age of 65 and more than 30% of the population over the age of 80.
Pathologically, Alzheimer's disease presents as extracellular amyloid plaques and intracellular neurofibrillary tangles. The neurofibrillary tangles are composed, e.g., of the microtubule-binding protein tau, which is assembled into paired helical and straight filaments. It has been suggested that these entities may be functionally linked, although the mechanisms by which amyloid deposition promotes pathological tau filament assembly is not clear.
The common denominator of intracellular neurofibrillary structures (neurofibrillary tangles, dystrophic neurites, and neurophil threads) is paired helical filaments (PHFs). The major protein subunit of the PHFs is microtubule associated protein tau in abnormally hyperphosphorylated form (Grundke-Iqbal et al., 1986; Wischik et al., 1988 a,b). Neurons with neurofibrillary changes degenerate, and the degree of this degeneration directly correlates with the degree of dementia in the affected individuals (Blessed et al., 1968).
Normal tau is a microtubule associated protein that distributes mainly to axons. Tau protein takes part in modulating the assembly, spatial organization and behavior of microtubules (MT) in neurons and probably glial cell bodies (Drewes et al., 1998; Drubin and Kirschner, 1986; Lo-Presti et al., 1995). Tau proteins are encoded by a single gene located on chromosome 17, but are detected as multiple isoforms in tissue extracts from adult brains (Goedert et al., 1989; Himmler A., 1989; Kosik et al., 1989). Heterogeneity of tau proteins is in part due to alternative splicing, giving rise to six isoforms in the adult human brain. These distinct isoforms differ by the presence or absence of 29- or 58-amino acid inserts in the amino-terminal region and by the addition or deletion of a tandem repeat (which can be repeated either 3 or 4 times) in a carboxy-terminal region of tau referred to as microtubule binding domain. This region is composed of imperfect repeats of 31 or 32 amino acid residues. Referring to the longest human tau protein isoform, htau40, containing all inserts (441 amino acid long) in humans, the smallest tau isoform contains 352 amino acid residues with three tandem repeats in the MT-binding domain and no amino terminal inserts, whereas the largest isoform contains 441 residues with four repeats and both amino terminal inserts.
A number of neurological diseases are known to have filamentous cellular inclusions containing microtubule associated protein tau, e.g., Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD) and a group of related disorders collectively termed frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), amyotropic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (CJD), dementia pugilistica (DP), Gerstmann-Straussler-Scheinker disease (GSSD), Lewy body disease and Huntington disease (Dickinson et al., 1998; DiFiglia et al., 1997; Formo, 1986; Hirano and Zimmerman, 1962; Nishimura et al., 1995; Prusiner 1996; Reed et al., 1998; Roberts, 1998; Schmidt et al., 1996; Shankar et al., 1989; Spillantini et al., 1998). Although the etiology, clinical symptoms, pathologic findings and the biochemical composition of inclusions in these diseases are different, there is emerging evidence suggesting that the mechanisms involved in aggregation of normal cellular proteins to form various filamentous inclusions are comparable. It is believed, that an initial alteration in conformation of microtubule associated protein tau, that initiates generation of nuclei or seeds for filament assembly, is one of the key features. This process can be influenced by the posttranslational modification of normal proteins, by mutation or deletion of certain genes and by factors that bind normal proteins and thus alter their conformation.
The tau protein is very hydrophilic, and is one of the most soluble proteins known. It can be readily extracted from brain tissue or cultured cells. Therefore, the aggregation of tau protein in AD is highly suspicious. In comparison, filamentous tau extracted from Alzheimer's diseased brain tissues is relatively insoluble. Besides phosphorylation, insoluble and normal soluble tau differ in the extent of posttranslational modifications, which include glycosylation, glycation, ubiquitination and racemization (Kenessey et al., 1995; Ko et al., 1999; Mori et al., 1987; Wang et al., 1996; Yan et al., 1994).
It has previously been reported that tau in AD brain neurofibrillary deposits is truncated at its C-terminus at the glutamic acid residue Glu391 (Novak, et al., 1989; Novak, et al., 1993). Truncation of tau at Glu391 leads to AD-specific conformational changes that are recognized by the conformational antibody MN423 (Novak, et al., 1989; Novak, et al., 1993; Csokova, et al., 2006; Skrabana, et al., 2006; and Skrabana, et al., 2007).
The mechanism by which tau protein is modified to take part in filament formation in AD is unknown. Phosphorylation of tau affects the potential of tau to form aggregates, producing either stimulatory or inhibitory effects, presumably depending on the site of phosphorylation (Crowther et al., 1994; Schneider et al., 1999). Hyperphosphorylation of tau at many sites appears to precede assembly into filaments, based on findings in mouse lines expressing human tau with FTDP-17T mutations (Lewis et al. 2000; Allen et al. 2002). Many in vitro studies taken together suggest (a) that the microtubule binding domain is important for assembly of tau filaments; and (b) that formation of tau filaments requires conformational change(s) of tau. These studies also indicate that none of tau modifications described therein are alone capable to induce filamentous tau formations that correlate with clinical expression of Alzheimer's disease.
Asuni et al., “Immunotherapy Targeting Pathological Tau Conformers in a Tangle Mouse Model Reduces Brain Pathology with Associated Functional Improvements”, Journal of Neuroscience, 27 (34): 9115-9129 (Aug. 2, 2007) discussed a study in which they sought to determine the effectiveness of active immunization directed against phosphorylated tau conformers in the CNS by immunizing P301L mice with a phosphorylated tau epitope with subsequent analysis of tau pathology and associated functional impairments. They determined that active immunization with a phosphorylated tau epitope Tau 379-408 (P-Ser396,404) reduces aggregated tau in the brain and slows progression of the tangle-related behavioral pheno-type in the mice.
Gamblin et al. “Caspase Cleavage of Tau: Linking Amyloid and Neurofibrillary Tangles of Alzheimer's Disease”, PNAS Vol 100, No. 17, pp. 10032-10037 (Aug. 19, 2003), reported that tau is proteolized by multiple caspases at a highly conserved aspartate residue (Asp421) in its C terminus in vitro and in neurons treated with amyloid-β (Aβ1-42) peptide. Tau was reported to be rapidly cleaved at Asp421 in Aβ-treated neurons (within 2 hours), and its proteolysis appears to precede the nuclear events of apoptosis. Gamblin et al. also demonstrated that caspase cleavage of tau generates a truncated protein that lacks its C-terminal 20 amino acids and assembles more rapidly and more extensively into tau filaments in vitro than wild-type tau. Using a monoclonal antibody that specifically recognizes tau truncated at Asp421, Gamblin et al. showed that tau is proteolytically cleaved at this site in the fibrillar pathologies of AD brain, and suggested that Aβ peptides promote pathological tau filament assembly in neurons by triggering caspase cleavage of tau and generating a proteolytic product with enhanced polymerization kinetics.
Delobel et al., “Analysis of Tau Phosphorylation and Truncation in a Mouse Model of Human Tauopathy”, American Journal of Pathology, Vol. 172, No. 1, pp. 123-131 (January 2008), investigated the time course of the appearance of phosphorylated and truncated tau in the brain and spinal cord of mice transgenic for human P301S tau protein. They reported that soluble tau was strongly phosphorylated at 1 to 6 months, and low levels of phosphorylated, sarkosyl-insoluble tau were detected at 2 months with a steady increase up to 6 months of age. They further reported that tau truncated at D421 was detected at low levels in Tris-soluble and detergent soluble tau at 3-6 months of age. They concluded that the late appearance and low abundance of tau ending at D421 indicates that it is unlikely that truncation at this site is necessary for the assembly of tau into filaments.
Zhang et al., “Truncated Tau at D421 is Associated with Neurodegeneration and Tangle Formation in the Brain of Alzheimer Transgenic Models”, Acta Neuropathol 117:687-697 (2009), analyzed spatial relationships among tau truncation, tau phosphorylation and neurodegeneration or tangle formation in a tau P302L mice and in a triple transgenic mouse model that produces both amyloid plaques and neurofibrillary tangles. They reported that a few neurons were detected that contained abundant truncated tau but were lacking hyperphosphorylation, and these neurons exhibited nuclear condensation, while truncated tau was commonly associated with high immunoreactivity of hyperphosphorylated tau and dense Gallyas silver staining. They concluded that tau truncation appears after tau hyperphosphyorylation in the brain of these two transgenic mouse models, and that accumulation of truncated tau, in the absence or the presence of physphorylated tau, is closely associated with a subset of neurons undergoing degeneration or containing neurofibrillary tangles.
Likewise, Khurana, et al. (2010), “Lysosomal Dysfunction Promotes Cleavage and Neurotoxicity of Tau InVivo”, PLoS Genet 6(7): e1001026. doi:10.1371/journal.pgen.1001026, demonstrated that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. They concluded that caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in AD.
Sigurdsson, “Tau-Focused Immunotherapy for Alzheimer's Disease and Related Tauopathies”, Current Alzheimer Research, Vo. 6, pp. 446-450 (2009), immunized transgenic mice expressing the P301L tau mutation with a 30 amino acid tau fragment that contained two phosphorylation sites that are prominent in AD (Tau 379-408[P-Ser396,404] and found that active immunization targeting this AD phospho-tau epitope reduces aggregated tau in the brain and prevents/slows progression of the tangle-related behavioral phenotype, including cognitive impairment. He concluded that these antibodies enter the brain and bind to pathological tau within neurons although the therapeutic effect may be at least in part due to clearance of extracellular tau that may have biological effects.
Calignon et al., “Caspase Activation Precedes and Leads to Tangles”, Nature Vol. 464/22, pp. 1201-1205 (April 2010), using in vivo multiphoton imaging to observe tangles and activation of executioner caspases in living tau transgenic mice (Tg4510 strain), found that caspase activation occurs first, and precedes tangle formation by hours to days. Based on this data, Calignon et al. proposed that caspase activation cleaves tau to initiate tangle formation, then truncated tau recruits normal tau to misfold and form tangles. They further suggested that tangles are “off pathway” to acute neuronal death, and that soluble tau rather than fibrillar tau may be the critical toxic moiety underlying neurodegeneration.
Kovacech et al., “Tau Truncation is a Productive Posttranslational Modification of Neurofibrillary Degeneration in Alzheimer's Disease”, Current Alzheimer Research, Vol 7, pp. 708-716 (2010), conclude that two posttranslational modifications of tau found in AD are assumed to play an inducing role in the neurofibrillary degeneration; truncation and hyperphosphorylation, and that it is impossible to precisely determine the temporal role of phosphorylation in the development of tau pathology because tau mutations are known to alter the conformation of the protein and lead to its higher and faster phosphorylation in vitro.
Horowitz et al., “Early N-Terminal Changes and Caspase-6 Cleavage of Tau in Alzheimer's Disease”, The Journal of Neuroscience, 24(36), pp. 7895-7902 (2004), reported immunohistochemical staining in a cohort of 35 cases ranging from noncognitively impaired to early AD with a panel of three N-terminal anti-tau antibodies: Tau-12, 5A6, and 9G3-pY18. Of these three, the phosphorylation-independent epitope of 5A6 was the earliest to emerge in the pathological lesions of tau, followed by the appearance of the Tau-12 epitope. It was reported that the unmasking of the Tau-12 epitope in more mature 5A6-positive tangles was not correlated with tau phosphorylation at tyrosine 18 (9G3-pY18). The extreme N-terminus of tau was lost later in the course of tangle evolution, correlating temporally with the appearance of a C-terminal caspase-truncated epitope lacking residues 422-441. In addition, caspase-6 cleaved the N terminus of tau in vitro, preventing immunoreactivity with both Tau-12 and 5A6, with the in vitro caspase-6 truncation site being identified as Asp13. The authors concluded that their results suggested a role for caspase-6 and N-terminal truncation of tau during neurofibrillary tangle evolution and the progression of AD.
It would be desirable to provide treatments which could interfere in the initiation of tau changes leading to filament formation or which could interfere with filament formation leading to tangles in disease conditions such as AD, and to develop therapeutic agents and dosage forms to treat, prevent or interfere in the progression of tauopathies.
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